ABSTRACT
Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P<0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system.
Subject(s)
Gastropoda , Hepatopancreas/metabolism , Muramidase/genetics , Schistosoma japonicum/immunology , Schistosomiasis japonica/immunology , Amino Acid Sequence , Animals , Bacteria/metabolism , Biological Evolution , Cloning, Molecular , Conserved Sequence/genetics , DNA, Complementary/analysis , Hepatopancreas/immunology , Hepatopancreas/parasitology , Host-Parasite Interactions/genetics , Molecular Sequence Data , Muramidase/immunology , Muramidase/metabolism , Schistosoma japonicum/pathogenicity , Transgenes/geneticsABSTRACT
BACKGROUND: Chemokines, a group of small and structurally related proteins, mediate chemotaxis of various cell types via chemokine receptors. In mammals, seven different CXC chemokine receptors denoted as CXCR1 to CXCR7 have been reported. However, the chemokine receptor CXCR5 has not been reported in other vertebrates. RESULTS: In the present study, the genomic sequence of CXCR5 was isolated from the grass carp Ctenopharyngodon idella. The cDNA sequence of grass carp CXCR5 (gcCXCR5) consists of 1518 bp with a 43 bp 5' untranslated region (UTR) and a 332 bp 3' UTR, with an open reading frame of 1143 bp encoding 381 amino acids which are predicted to have seven transmembrane helices. The characteristic residues (DRYLAIVHA) and conserved cysteine residues are located in the extracellular regions and in the third to seventh transmembrane domains. The deduced amino acid sequence shows 37.6-66.6% identities with CXCR5 of mammals, avian and other fish species. The grass carp gene consists of two exons, with one intervening intron, spaced over 2081 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcCXCR5 is clustered with those in other teleost fish and then in chicken and mammals. Real-time PCR analysis showed that gcCXCR5 was expressed in all tested organs/tissues and its expression level was the highest in trunk kidney, followed by in the spleen. The expression of gcCXCR5 was significantly modulated by immunostimulants such as peptidoglycan (PGN), lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C) and phytohaemagglutinin (PHA). CONCLUSION: The cDNA and genomic sequences of CXCR5 have been successfully characterized in a teleost fish, the grass carp. The CXCR5 has in general a constitutive expression in organs/tissues examined, whereas its expression was significantly up-regulated in immune organs and down-regulated in brain, indicating its potential role in immune response and central nervous system.
Subject(s)
Carps/genetics , Gene Expression Regulation , Receptors, CXCR5/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/genetics , Exons/genetics , Gene Expression Profiling , Genome/genetics , Introns/genetics , Mammals/genetics , Molecular Sequence Data , Phylogeny , Receptors, CXCR5/chemistry , Receptors, CXCR5/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
The interferon regulatory factor 5 (IRF-5) is known to be involved in the innate immune response and in the regulation of DNA damage-induced apoptosis. In the present study, the cDNA and genomic sequences of IRF-5 were identified in grass carp (Ctenopharyngodon idella). The cDNA of grass carp IRF-5 (gcIRF-5) contains an open reading frame (ORF) of 1560 nucleotides, encoding a putative 519 amino acid protein, which showed 34.5-83.9% identity to IRF-5 homologues from mammals, amphibian, avian and fish, and 96.2% and 95.0% identity to zebrafish IRF-5 in the DNA-binding domain (DBD) and IRF association domain (IAD), respectively. The genomic DNA sequence of gcIRF-5 contains 6075bp consisting of 9 exons and 8 introns. The expression of gcIRF-5 was observed in all organs examined. The analysis of real-time quantitative RT-PCR revealed that grass carp reovirus (GCRV) induced the expression of gcIRF-5 in spleen and head kidney.
